This invention relates to an analytical method and an analytical apparatus for determining fluorescence or phosphorescence. More particularly, it relates to a method and an apparatus suitable for allowing a sample to react in a cuvette or a reaction container and then determining fluorescence or phosphorescence resulting from the reaction.
In recent years, there has been a strong demand in the field of medical treatment for quantitative determination of a very small amount of substances. There have already been devised various methods of quantitatively determining substances in body fluids by immunological technique, namely by utilizing antigen-antibody reactions. For example, there have been known methods of measuring the sedimentation or the agglomeration of reactants, such as the capillary sedimentation method, immunoturbidimetry, immunonepherometry and the latex agglomeration method, and labelled immunoassay using labelling substances such as enzymes, radioisotopes or fluorescent substances. Among these methods of determination, labelled immunoassay is attracting attention owing to such factors as high sensitivity in determination and ease in handling the necessary reagents. Labelled immunoassays include those of a homogeneous reaction system (homogeneous immunoassay) and those of a heterogeneous reactio system (heterogeneous immunoassay). A homogeneous reaction system is a system in which the separation of a marker substance bound to the reaction product of an antigen and an antibody (B: BOUND) from the marker substance present in the free state (F: FREE), namely B/F separation, is unnecessary and the determination can be conducted while the above two are in a mixed state, whereas a heterogeneous reaction system is a system in which the two are first separated from each other and then the label amount of either of the two is determined.
When the concentration of a target substance in sample is determined by a fluorescently labelled method using a homogeneous reaction system, there arises a problem of blank fluorescence. This point will be explained a little more in detail below.
Conventional fluorophotometers basically consist of five parts of a light source, an excitation wavelength selector such as a monochromator, a sample chamber, an emission wavelength selector or a monochromator, and a photodetector. Thus, a light radiated from a light source is separated into its spectral components through an excitation wavelength selector and then irradiated to a sample. The resulting fluorescence is taken out from a direction perpendicular to the direction of irradiated light, passed through a fluorescence wavelength selector, and then received by a detector. The light arriving at the detector has much possibility of being contaminated with other components than the fluorescence originating from fluorescent molecules. These superfluous lights are called blank fluorescence. Conceivable causes of blank fluorescence include scattered or reflected light, Raman scattering light from solvents, fluorescence from solvents and fluorescence cells, the secondary light of scattered or reflected light, and a component of abnormal reflection occurring in the optical system including a spectroscope.
It is needless to say that when the fluorescent sample is in a substantially high concentration the intensity of these blank fluorescence components becomes relatively weak. However, when the concentration is extremely low (for instance, in the determination of ultramicroquantity components) or when the sample in turbid, the influence of the blank fluorescence components on the determination of the fluorescence component originally intended is large and cannot be neglected. Further, fluorescence originating from the contaminants in the sample also causes an error in determination. Blank fluorescence components which appear at the same wavelength as that of exciting light come mainly from light scattered by solvent molecules as Reyleigh scattering.
Further, the proteinous reactant in an agglomerated state formed as the result of an antigen-antibody reaction appears itself in a high concentration and inevitably causes the increase of scattered light. When the resolving power of the spectroscope is poor, the bottom of the scattered light component extends toward long wavelength side and overlaps extensively with the fluorescence emission region. Although this problem is somewhat alleviated when the slit width of the spectroscope is reduced in determination, then the detected intensity of fluorescence is also reduced and a determination of high accuracy cannot be expected.
A method of decreasing the influence of blank fluorescence is disclosed, for example, in U.S. Pat. No. 4,421,860. In the disclosed method, fluorescence resulting from a free, fluorescently-labelled reactant on one hand and fluorescence resulting from the fluorescently labelled reactant bound to giant particles on the other hand can be determined separately from each other; however, since the sample is not moved as to its position, this method is not suited to continuous analysis.
Blank fluorescence and fluorescence originating from contaminating components are hereinafter referred to collectively as background fluorescence.